Abstract
BACKGROUND: Regular use of high-concentrate diets can improve the growth of animals, especially high-yielding cattle and sheep. However, high-concentrate diets can alter rumen fermentation and microbial community structure, leading to rumen acidosis. Therefore, this study aimed to investigate the effects of 5,6-dimethylbenzimidazole (5,6-DMB) and cobalt supplementation in high-concentrate diets on rumen fermentation and microorganisms and ruminal fluid metabolome in sheep. METHODS: Twelve rumen-fistulated Kazakh rams (age: 8 months; average initial body weight [BW]: 39.23 ± 2.61 kg) were randomly allocated to two groups (n = 6 rams/group): control (CON) and DMB. Sheep in the CON group were fed a basal diet (concentrate-to-forage ratio of 70:30), whereas those in the DMB group were fed a basal diet supplemented with 100 mg/d 5,6-DMB (analytical purity ≥ 99%) and 0.5 mg/kg cobalt (cobalt chloride as supplementary Co, zeolite powder as carrier; Co content ≥ 1%). Notably, the study lasted for 32 days (adaptation period: 1-14 days; experimental period: 15-32 days). During the experiment, feed samples and residual feed were collected, weighed, and recorded. Rumen fluid samples were collected at 0 h before morning feeding and at 1, 3, 5, and 7 h after feeding on days 29-32 of the experiment to measure ruminal pH. RESULTS: Ruminal pH was significantly higher (P < 0.01) in the DMB group than in the CON group at 1, 3, 5, and 7 h after feeding. Microbiota analysis showed that the dominant phyla in the two groups were Firmicutes (46.70%), Bacteroidetes (43.79%), and Proteobacteria (2.66%). Additionally, the dominant genera were Prevotella (14.78%), Quinella (7.47%), Succiniclasticum (7.40%), Rikenellaceae_RC9_gut_group (4.44%), NK4A214_group (3.89%), and Ruminococcus (3.67%). Alpha diversity analysis showed that Simpson and Shannon indices were significantly higher (P < 0.05) in the DMB group than in the CON group. Metabolome analysis identified 20 significantly different metabolites between the two groups, including 12 positive ions and 8 negative ions. Pathway enrichment analysis showed that the metabolites were mainly enriched in 16 differential metabolic pathways, including fatty acid metabolism and tricarboxylic acid cycle. Correlation analysis revealed an association between ruminal microorganisms and metabolites (propionate, L-malic acid, and arginine). CONCLUSIONS: Supplementing high-concentrate diets with 5,6-DMB (100 mg/d) and cobalt (0.5 mg/kg) increased ruminal pH, ammonia nitrogen concentration, and propionate concentration to a certain extent, reduced lactate concentration and lactate dehydrogenase activity, regulated ruminal microbial community structure (Quinella and Succinivibrio), and improved rumen metabolite levels (L-malate, pyruvate, and acetate concentrations). Conclusively, these findings suggest that supplementing high-concentrate diets with 5,6-DMB and cobalt may promote vitamin B12 synthesis and improve energy utilization efficiency.